Disinfectant compositions comprising an orange oil mixture and methods of use thereof

ABSTRACT

The present invention discloses a highly potent, non-toxic, disinfectant that can be used for a wide breadth of applications. The disinfectant comprises hydrogen peroxide (H 2 O 2 ), orange terpene oil, orange valencia oil, a non-ionic emulsifier (polysorbate 80), and water (H 2 O). Applications of the disinfectant include, but are not limited to, uses as a mouthwash, skin cleanser, or as a germicidal for disinfecting surfaces such as foodstuff, plant matter, leather, wood, metal, plastic and fabrics. Methods for using the disinfectant composition of the invention are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit under 35 U.S.C § 119(e) of U.S.Provisional Application No. 60/720,811 filed Sep. 27, 2005.

FIELD OF INVENTION

The present invention relates generally to an environmentally friendly,non-toxic, food grade, disinfectant composition that can be used formultiple applications including, but not limited to, uses as amouthwash, skin cleanser, or as a germicidal for disinfecting surfacessuch as foodstuff, plant matter, leather, wood, metal, plastic andfabrics.

BACKGROUND OF THE INVENTION

A number of products have been developed for the purpose of disinfectingand cleaning. However, many of these products use toxic, poisonouschemicals that are detrimental to the environment and to our health.

Hydrogen peroxide is a strong, environmentally friendly, disinfectantwith a broad spectrum of antimicrobial activity that has been widelyused in the healthcare field. Unfortunately, hydrogen peroxide is also astrong oxidizing agent that in high concentrations can damage tissue anddamage surfaces such as foodstuff, making them more vulnerable topathogenic penetration.

Essential oils, are natural products known to have antimicrobialproperties and disinfectant solutions based on essential oils have beenformulated, U.S. Pat. No. 6,846,498. However, the potency and spectrumof action of many of these formulations lag behind those exhibited byother antimicrobials. Furthermore, formulations are difficult to makebecause the oils are not readily miscible in water.

SUMMARY OF INVENTION

The present invention is directed to disinfectant compositions and theiruse.

The invention provides a disinfectant composition comprising hydrogenperoxide (H₂O₂), orange terpene oil, orange valencia oil, a non-ionicemulsifier (e.g. polysorbate 80), and deionized or distilled water(H₂O). The composition can include varied amounts of each of theseingredients.

In one embodiment, the orange terpene oil is present in the compositionfrom 5% to 40% v/v, the orange valencia oil is present in thecomposition from 5% to 40% v/v, the non-ionic emulsifier is present inthe composition from 5% to 50% v/v, the distilled or deionized H₂O ispresent in the composition from 5% to 80% v/v and the hydrogen peroxideis present in the composition from 1.5% to 8% H₂O₂ w/v In anotherembodiment, the composition comprises one half as much non-ionicemulsifier (e.g. polysorbate) by volume as the combined volume of orangevalencia oil and orange terpene oil.

In one preferred embodiment the disinfectant composition comprises 5.25%H₂O₂ (15% of a 35% hydrogen peroxide solution), 10% v/v orange terpeneoil, 5% v/v orange valencia oil, 10% v/v polysorbate 80, and 60% v/vdistilled water.

In one embodiment, the disinfectant composition further comprises anantioxidant preservative of H₂O₂ such as oil of rosemary.

In one embodiment, the composition further comprises antimicrobials suchas quaternary ammonium compounds, triclosan, cetyl pyridium chloride,domiphen bromide, zinc compounds, sanguinanine soluble pyrophosphates,fluorides, alexidine, octonidine, and EDTA. In addition, thedisinfectant composition can also comprise surfactants, common buildersthat improve surfactant effectiveness, saponifiers, chelating agents,and/or other solvents.

The invention provides a method for disinfecting surfaces comprisingapplying the composition to the surface.

The invention further provides a method for reducing the number of moldspores on a surface. The method comprises contacting the surfacecontaining the mold spores with the composition.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a chart summarizing the results of an EPA acute oraltoxicity test where three female Sprague-Dawley rats received an orallimit dose of 50000 mg/kg of a germicidal spray comprising 60% distilledwater, 10% polysorbate 80, 5% orange valencia oil, 10% orange terpeneoil, and 15% of a 35% hydrogen peroxide solution. Time after dosing,mortality and clinical signs are indicated.

FIG. 2 shows a chart summarizing the results of an EPA primary eyeirritation test where the eyes of six New Zeland Rabbits were exposed toa germicidal spray comprising 60% distilled water, 10% polysorbate 80,5% orange valencia oil, 10% orange terpene oil, and 15% of a 35%hydrogen peroxide solution. Various times after dosing, the L (left) andR (right) eye were given a score as indicated in FIG. 3.

FIG. 3 shows a chart referencing the scale used for scoring ocularlesions that are scored in the primary eye irritation test describedherein.

FIG. 4 shows a chart referencing the scale used for scoring skinreactions in the primary dermal irritation test described herein.

FIG. 5 shows a chart summarizing the results of a EPA primary dermalirritation test where the skin of three New Zeland Rabbits were exposedto a germicidal spray comprising 60% distilled water, 10% polysorbate80, 5% orange valencia oil, 10% orange terpene oil, and 15% of a 35%hydrogen peroxide solution. Effects of exposure were given a score asindicated in FIG. 4.

FIG. 6 shows a chart of the results of a Virucidal Efficacy Testindicating the measured titer of Avian Influenza Virus after exposure toa germicidal spray comprising 60% distilled water, 10% polysorbate 80,5% orange valencia oil, 10% orange terpene oil, and 15% of a 35%hydrogen peroxide solution; lot 14-G-07 and lot 14-G-03, as described inExample V. (----) lines indicate that no virus was detected.

FIGS. 7A-7C show charts of the results of control experiments performedin the Virucidal Efficacy Test described in Example V. FIG. 7A shows theresults of neutralizer effectiveness and toxicity related controls. FIG.7B shows the plate recovery control, column titer control and virusstock titer control. FIG. 7C shows control results for the hostviability control. For FIGS. 7A-7C; (0)=no toxicity observed. (+)=AvianInfluenza A virus was detected, hemagglutination observed, (−)=AvianInfluenza A virus was not detected, No hemagglutination observed,PNS=Post neutralized sample, ND=Not determined.

DETAILED DESCRIPTION

We have discovered a highly potent non-toxic, environmentally friendly,disinfectant that can be used for a wide breadth of applications. Thedisinfectant described herein is composed of food grade material andcomprises hydrogen peroxide (H₂O₂), orange terpene oil, orange valenciaoil, a non-ionic emulsifier (e.g. polysorbate 80), and distilled ordeionized water (H₂O). The disinfectant is very potent and, as shown inExample 1, highly effective at killing microorganisms including, but notlimited to, bacteria (gram positive or gram negative), bacterial spores,molds, fungi and viruses such as Salmonella cholerasuis, Staphylococcusaureaus, Pseudomonas aeruginosa, Escherichia coli, Streptococcuspneumonia, and Listeria monocytogenes, and Influenza virus.

An advantage of the disinfectant composition of the invention over otherdisinfectants is that it can be composed of entirely food gradematerials while maintaining its high degree of potency against bacteriaand other microorganisms. The non-toxic components of the compositiondescribed herein work synergistically against microorganisms. Not to bebound by theory, it is believed that the orange valencia oil togetherwith the other components weakens the microrganisms(bacteria/fungus/mold spores/viruses) making them more vunerable to theH₂O₂.

In one embodiment, the disinfectant composition further comprises oil ofrosemary, e.g. CAS Number 8000-25-7, which can be obtained from PolaromeInternational, Inc. (200 Theodore Conrad Drive, Jersey City, N.J.07035).

Any non-ionic emulsifier can be used in the disinfectant composition ofthe invention, including, but not limited to, alkyl polyethyleneoxyethers, alkyl phenol polyethyleneoxy ethers, polyethyleneoxy amines,polyethyleneoxy fatty acids, and alkyl dimethyl amino oxides. In anotherembodiment, the composition comprising hydrogen peroxide (H₂O₂), orangeterpene oil, orange valencia oil, a non-ionic emulsifier, and distilledor deionized water (H₂O) does not comprise an anionic surfactant or anamphoteric surfactant.

In one embodiment, the non-ionic emulsifier of the disinfectantcomposition is polysorbate 80 (CAS Number 9005-65-6).

In one embodiment, the disinfectant is a composition that comprises 60%v/v distilled water, 10% v/v polysorbate 80, 5% v/v orange valencia oil,10% v/v orange terpene oil, and 15% v/v of a 35% wt. % hydrogen peroxidesolution (equivalent to 5.35% of H₂O₂). The composition can be used atfull strength or can be diluted with water. Dilutions can range from 1:1to 1:10,000.

The disinfectant composition can be formulated, if desired, as a gel,spray, foam, or paste, or as a disinfecting wipe, using standardformulations known in the art as appropriate.

The disinfectant composition can be made by mixing the componentstogether using any known means. Preferably the components are mixedtogether sequentially. For example, the components are added in sequenceto distilled water in the following order: orange terpene oil, orangevalencia, then polysorbate 80. While being mixed at moderate speed,hydrogen peroxide is then added and mixed for approximately fiveminutes. In one preferred embodiment, the distilled water is keptbetween 90° and 100° Fahrenheit to facilitate the emulsificationprocess.

The components can be obtained from any source. Preferably food gradecomponents are used. As an example source, the orange terpene oil andorange valencia oil can be obtained from Polarome International, Inc.(200 Theodore Conrad Drive, Jersey City, N.J. 07035) (Orange Terpenes,CAS Number 68647-72-3, EINECS Number 232-433-8; Orange Oil Valencias,CAS Number 8008-57-9, EINECS Number 232-433-8)); the polysorbate 80 canbe obtained from Spectrum Chemicals (14422 South San Pedro Street,Gardena, Calif. 90248) and the hydrogen peroxide from Solvay ChemicalsInc. (1632 Haden Road, Houston, Tex. 77015) or from FMC corporation(1735 Market Street Philadelphia, Pa 19103), (Durox®35%). The orangevalencia oil can be derived from a cold press expression method whichpreserves viable anti-oxidants. In one embodiment, the orange valenciaoil has at least 1.4% aldehydes.

While it is preferable to use only food grade components in thecomposition, other non-food grade components can also be added.

The composition described herein can include multiple surfactants,including, but not limited to nonionic surfactants such as nonylphenolethoxylate, alcohol ethoxylates, octylphenol ethoxylate, coconutdiethanolamide (cocoamide DEA), unspecified nonionic surfactant; anionicsurfactants such as linear alkylbenzene sulfonate (dodecylbenzenesulfonate), alcohol sulfates (lauryl sulfates), alcohol ether sulfates(lauryl ether sulfates, laureth sulfates), sodium alkyl polyethersulfonate, alkyl polyglycosides, unspecified anionic surfactant, andsoap; amphoteric surfactants such as, alkylbetaine, unspecifiedamphoteric surfactant; and cationic surfactants such as alkyl dimethylbenzyl ammonium chlorides, unspecified quaternary ammonium chlorides orcompounds, alkylaryl dimethyl ammonium chloride, dimethyl ethyl benzylammonium chloride, ethylbenzene ammonium chloride, didecyl dimethylammonium chloride, octyl dimethyl ammonium chloride.

The composition of the invention can further comprise common buildersthat improve surfactant effectiveness, saponifiers, chelating agents,and/or other solvents, examples of such additives include, but are notlimited to, acetic acid, hydrochloric acid, citric acid, sodiumhydroxide, potassium hydroxide, carbonates sodium carbonate, sodiumbicarbonate, pyrophosphates, polyphosphates, phosphate esters,orthophosphates, sodium metasilicate, sodium silicate, ethanolamines,carbonates, silicates, EDTA, STPP, and zeolites/PCA, isopropanol,methanol, ethanol, 2-butoxyethanol, diethylene glycol ethyl ether,diethylene glycol, monomethylether, 1-methoxy-2-propanol,2-2-butoxyethyoxyethanol, d-limonene, pine oil, tall oil, ammonia(ammonium hydroxide), hydrocarbons, propylene glycol, ethylene glycol,or 1,3-proponediol.

Although not necessary, it is possible to employ other antimicrobialagents in the composition of this invention, for example quaternaryammonium compounds, phenols, alcohols, sodium hypochlorite, pine oil orother known antimicrobial oils. Examples of quaternary ammoniumcompounds include, but are not limited to alkyl dimethyl benzyl ammoniumchlorides, unspecified quaternary ammonium chlorides or compounds,alkylaryl dimethyl ammonium chloride, dimethyl ethyl benzyl ammoniumchloride, ethylbenzene ammonium chloride, didecyl dimethyl ammoniumchloride, and octyl dimethyl ammonium chloride. Preferably thequaternary ammonium compound is present in the composition at 0.01-1%.Example phenols include, but are not limited to ortho-benzylparachlorophenol, ortho-phenylphenol, and para-tertiary-amylphenol.Preferably the phenol is present in the composition at 2-5%. Examplealcohols include but are not limited to Isopropyl alcohol and ethanol.Preferably, sodium hypochlorite is present in the composition from0.5-5%. Other exemplary antimicrobial agents include, but are notlimited to, triclosan, cetyl pyridium chloride, domiphen bromide, zinccompounds, sanguinanine soluble pyrophosphates, fluorides, alexidine,octonidine, EDTA, and the like.

The non-toxic nature of the disinfectant described herein allows for itsuse not only in applications where the harshness of the disinfectant isirrelevant, but also in applications more sensitive in nature, forexample when disinfecting skin, treating plants and food stuff, andkilling oral microorganisms.

In one embodiment, the disinfectant described herein is used to sanitizeporous or non-porous surfaces. Any surface can be cleaned using thedisinfectant of the invention including, but not limited to, leather,wood, metal, plastic, synthetics, and fabrics.

The composition of the invention can disinfect surfaces containingbacteria, bacterial spores fungus, and/or viruses (DNA or RNA viruses).

The compositions can be used to inactivate vegetative bacteria andbacterial spores upon contact. Bacteria that can be inactivated by thecompositions can be gram negative or gram positive bacteria. gramnegative bacteria include, for example and without limitation, Vibrio,Salmonella, Shigella, pseudomonas, Escherichia, Klebsiella, Proteus,Enterobacter, Serratia, Moraxella, Legionella, Bordetella, Gardnerella,Haemophilus, Neisseria, Brucella, Yersinia, Pasteurella, Bacteroids, andHelicobacter gram positive bacteria include, for example, and withoutlimitation, Bacillus, Clostridium, Arthrobacter, Micrococcus,Staphylococcus, Streptococcus, Listeria, Corynebacteria, Planococcus,Mycobacterium, Nocardia, Rhodococcus, Andacidfast bacilli such asMycobacterium. In one embodiment the compositions can be used toinactivate bacillus, including, without limitation B. anthracis, B.cereus, B. circulans, B. subtilis, and B. megaterium. Compositions ofthe invention can also be used to inactivate Clostridium, e. g., C.botulinum, C. perfringens, and C. tetani. Other bacteria that can beinactivated by the composition include, but are not limited to, H.influenzae, N. gonorrhoeae, S. agalactiae, S. pneumonia, S. pyogenes andV. cholerae.

Contacting a virus with the composition of the invention can inactivatea virus.

The effect of compositions on viral agents can be monitored using anysuitable means, such as, for example, plaque reduction assay (PRA),cellular enzyme-linked immunosorbent assay (ELISA), P-galactosidaseassay, and electron microscopy (EM). Viruses which can be inactivated bycontact with the composition include, without limitation, virus of thefamilies Baculoviridae, Herpesviridae, Iridoviridae, Poxviridae,“African Swine Fever Viruses,” Adenoviridae, Caulimoviridae, Myoviridae,Phycodnaviridae, Tectiviridae, Papovaviridae, Circoviridae,Parvoviridae, Hepadnaviridae,Cystoviridae,Bimaviridae, Reoviridae,Coronaviridae, Flaviviridae, Togaviridae, “Arterivirus,” Astroviridae,Caliciviridae,Picornaviridae, Potyviridae, Retroviridae,Orthomyxoviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae,Arenaviridae, and Bunyaviridae.

In one embodiment, the virus is herpes, pox, papilloma, corona,influenza, hepatitis, sendai, sindbis and vaccinia viruses, West Nile,hanta, and viruses which cause the common cold.

In yet another embodiment, contacting a fungus with the composition ofthe invention inactivates the fungus. In one embodiment, the fungus is ayeast, such as, for example various species of Candida (e. g., Candidaalbicans) or filamentous yeast including but not limited to Aspergillusspecies or dermatophytes such as Trichophyton ubrura, Trichophytonmentagrophytes, Microsporum canis, Microsporum gypseux, andEpiderophytonfloccosum, and types thereof, as well as others.

The composition of the invention can also be used for mold remediationfor building, equipment, and facilities. Examples of molds include, butare not limited to Cladosporium, Fusarium, Alternaria, Curvularia,Aspergillus, and Penicillium.

The disinfectant described herein can also be used as an aerosol orspray to eliminate odors, e.g. as a room or fabric deodorizer.

In one embodiment, the disinfectant described herein is used as amouthwash to eliminate bad breath and to reduce the presence of oralmicroorganisms. Although not necessary, additional conventionalcomponents may be added to the disinfectant of the invention as inmouthwashes of the prior art. For example, softeners such as glycerinmay be added to enhance the lubricous mouth feel of the mouthwash as itis used and to provide a refreshing, moist, organoleptic feelingthereafter. Glycerin may be incorporated in amounts of from about 0.05%w/v to about 10.0% w/v, and preferably in an amount of about 7.5% w/v.Sweeteners such as aspartame or sodium saccharin and the like may beadded for better taste in amounts of from about 0.005% w/v to about 1.0%w/v, and preferably in an amount of approximately 0.05% w/v. Otheressential oils can be added to alter the flavor. Zinc chloride or otherzinc salts e.g. zinc gluconate, zinc sulfate etc., may be added as anastringent for an “antiseptic cleaning” feeling in an amount of fromabout 0.0025% w/v to about 0.200% w/v.

In one embodiment, the disinfectant described herein is used in medicalapplications, such as to clean skin wounds, or skin surfaces. Thedisinfectant can also be used as a hand sanitizer. The disinfectant isvery potent and highly effective at killing microorganisms including,but not limited, to Salmonella cholerasuis, Staphylococcus aureaus,Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumonia,Listeria monocytogenes and influenza.

In one embodiment, the disinfectant described herein is used asgermicidal for disinfecting/cleaning foodstuff or plant matter. Hydrogenperoxide solutions have also been found to be effective at inhibitingsprouting and rooting of foodstuffs, such as potatoes or othervegetables, thereby extending the shelf life of perishable food stuffsand plant matter. See for example, U.S. Pat. No. 6,348,187 which isherein incorporated by reference. The disinfectant composition can besprayed directly on plant matter or mixed with soil to discourageinfestation with parasitic organisms.

In one embodiment, the composition is used in the food industry inpreventing and treating food contaminated with pathogens. Thus, suchcompositions may be used to reduce or inhibit microbial growth orotherwise abrogate the deleterious effects of microbial contamination offood. For example, the composition can be used to kill bacteria andfungus on poultry eggs, fruit, vegetables, and meat. Also, the inclusionof the compositions of the invention within the food product itselfwould be effective in killing bacteria that may have been accidentallycontaminated meat or poultry. The composition can be included in juiceproducts to prevent growth of certain fungi, which cause contaminationand lead to production of mycotoxins. For these applications, thecompositions is applied in food industry acceptable forms such aswashes, dips, additives, preservatives, or seasonings. The use of mediaand agents for additives, preservatives, and seasonings that areacceptable in food industry is well known in the art. Except insofar asany conventional additives, preservatives and seasonings areincompatible with the disinfectant composition of the invention, theiruse in preventing or treating food born microbes and their toxicproducts is contemplated. Supplementary active ingredients may also beincorporated into the compositions.

In one embodiment the disinfectant is loaded onto a cleaning wipe. Thecleaning wipe, upon which the disinfectant composition is loadedthereon, is made of an absorbent/adsorbent material. Typically, thecleaning wipe has at least one layer of nonwoven material. Nonlimitingexamples of commercially available cleaning wipes that can be usedinclude DuPont 8838, Dexter Z A, Dexter 10180, Dexter M10201, Dexter8589, Ft. James 836, and Concert STD60LN, and Ahlstrom 4759. All ofthese cleaning wipes include a blend of polyester and wood pulp. DexterM10201 also includes rayon, a wood pulp derivative. The loading ratio ofthe cleaning composition onto the cleaning wipe is about 2-5:1, andtypically about 3-4:1. The disinfectant composition is loaded onto thecleaning wipe in any number of manufacturing methods. Typically, thecleaning wipe is soaked in the disinfectant composition for a period oftime until the desired amount of loading is achieved.

In one embodiment, the disinfectant composition is packaged in apressurized gas aerosol can. Common aerosol propellants include butane,isobutane, liquefied natural gas, and propane.

The invention provides methods for disinfecting surfaces to inactivatepathogenic organisms comprising contacting a surface with thedisinfectant composition of the invention. The step of contacting caninvolve contacting any substrate, which may be or is suspected to becontaminated, with the composition of the invention. By substrate it ismeant, without limitation any subject, such as a human or an animal(contact can be in vivo or ex vivo, any article, any surface, or anyenclosure. A pathogenic microorganism can be, without limitation, abacteria, a virus, a fungus, a protozoan or a combination thereof.

The step of contacting can be performed for any amount of timesufficient to inactivate a microorganism. In one embodiment,inactivation occurs within about 5 minutes to about 10 minutes afterinitial contact. However, it is understood that when the emulsions areused in a therapeutic context and applied topically or systemically, theinactivation may occur over a longer period of time, for example, 5, 10,15, 20, 25, 30, 60 minutes or longer after administration.

The step of contacting can be performed using any appropriate means ofapplication. For example, compositions can be administered by spraying,fogging, misting, exposure to aerosols, wiping with a wet or saturatedcloth or towlette, drenching, immersing.

The invention further provides a method for reducing the number of moldspores on a surface. The method comprises contacting the mold sporeswith the composition comprising hydrogen peroxide (H₂O₂), orange terpeneoil, orange valencia oil, a non-ionic emulsifier (e.g. polysorbate 80),and distilled or deionized water (H₂O). Any mold can be treated usingmethods of the invention.

EXAMPLES Example I Efficacy

Methods

The germicidal spray composition comprising 60% distilled water, 10%polysorbate 80, 5% orange valencia oil, 10% orange terpene oil, and 15%of a 35% Hydrogen peroxide solution was tested for its ability to killSalmonella cholerasuis, Staphylococcus aureaus, Pseudomonas aeruginosa,Escherichia coli, Streptococcus pneumonia, and Listeria monocytogenes.Three lots were tested, one being at least greater than 60 days old.

The analysis was performed per “Germicidal Spray Test”, AOAC 17^(th)edition, 6.3.03, versus Salmonella cholerasuis ATCC 10708,Staphylococcus aureaus ATCC 6538, Pseudomonas aeruginosa ATCC 15442,Escherichia coli ATCC 8739, Streptococcus pneumonia ATCC 49619, andListeria monocytogenes ATCC 19113. Testing was performed as required bythe EPA FIFRA. The AOAC procedure used in this study is a widely knownand accepted method for disinfectant evaluation.

Microorganisms were grown for 48 h in nutrient broth. Glass slides werethen inoculated with the grown culture and the inoculated slides weresubsequently exposed to the germicidal spray for 10 minutes (60microorganism inoculated slides/lot/microorganism were tested). Afterexposure to the disinfectant, slides were used as inoculates in leethenmedia and microorganism growth determined. Positive, negative andinhibition controls were performed.

Results

All organisms tested were found to be susceptible to the disinfectant.Absolutely no growth was observed in media that was inoculated with theorganism slides which were exposed to disinfectant. Controls includeduninoculated containers of leethen broth (media controls) and sterileuninoculated glass slides (negative control), there was no growth; Glassslides inoculated with organism and a sterile glass slide immersed intest disinfectant (inhibition control), there was no growth whichindicates that the letheen broth neutralizes the disinfectant; Glassslide inoculated with organism (viability control), there was growth;and Glass slide inoculated with organism (enumerated), >10⁶ cfu.

Example II EPA Acute Oral Toxicity

Methods

The germicidal spray composition comprising 60% distilled water, 10%polysorbate 80, 5% orange valencia oil, 10% orange terpene oil, and 15%of a 35% Hydrogen peroxide was tested for toxicity according to protocolnumber X5D050G, which incorporates by reference Northview StandardOperating Procedure 16D-05 and is on file in Northview PacificLaboratories, Inc. No amendments were made to the protocol.

A limit screen test was performed using three female Sprague-Dawleyrats, which received an oral limit Dose of 50000 mg/kg of the testarticle. The animals were observed for mortality, weight change andtoxic signs for a two week period. Animals were observed daily andweighed on days 7 and 14.

Animal preparation. The animals were fasted overnight before doseadministration. During fasting they continued to receive water adlibitum. Food was withheld until four hours after dosing in order tofacilitate gastrointestinal absorption of the test article.

Sample preparation. The density of the test article was 1 g/ml andshaken before use.

Dosing procedure. The dose was administered by means of a gavage needleattached to a hypodermic syringe. The test animals received a 5 mL/kgsolution containing the test article. Three rats were dosed on the firstday of dosing. Because all three rats survived no further testing wasrequired.

Results

A single oral administration of germicidal spray product at a limit doseof 5000 mg/kg produced no mortalities. The clinical observations aresummarized in FIG. 1. All animals gained weight during the test periodand no abnormalities upon necropsy were observed.

Example III EPA Primary Eye Irritation Test

Method

The germicidal spray composition comprising 60% distilled water, 10%polysorbate 80, 5% orange valencia oil, 10% orange terpene oil, and 15%of a 35% Hydrogen peroxide was tested for toxicity according to protocolnumber X5D051G, which incorporates by reference Northview StandardOperating Procedure 16D-08 and is on file in Northview PacificLaboratories, Inc. No amendments were made to the protocol.

Six New Zeland Rabbits were used. On the day prior to dosing the rabbitseyes were examined using flourescein sodium opthamalic strips andultraviolet light. Only rabbits without eye defects or irritation wereused. A 100 ul volume of test material was introduced into theconjuctival sac of the right eye of each rabbit. The left eye remaineduntreated. The treated eye was washed out with physiological saline 24hours after dosing.

The eyes were examined and graded for ocular reaction at 1, 24, 48, and72 hours after application of the test substance for corneal ulcerationor opacity, inflammation of the iris, or redness and chemosis of theconjunctivae. The results are interpreted according to the Kay andCalandra Method (Kay J. H. & Calandra, J. C., “Interpretation of eyeirritation tests”, Journal of society of cosmetic chemists, 13:281-289,1962).

Results

Based on the Kay and Calandra method of classifying eye irritationproperties, the test article was determined to be moderately irritatingto eyes of New Zeland Rabbits.

All animals remained healthy throughout the study period. The primaryeye irritation scores are shown in FIG. 2. A table indicating the scalefor scoring the ocular lesions can be found in FIG. 3. There wassignificant irritation in all 3 animals beginning at the 1 hour scoringand continuing through the 96 hour scoring. The scores all went back to“0”, normal scoring, by the 7-day scoring.

Example IV EPA Primary Dermal Irritation Test

Method

The germicidal spray composition comprising 60% distilled water, 10%polysorbate 80, 5% orange valencia oil, 10% orange terpene oil, and 15%of a 35% Hydrogen peroxide was tested for toxicity according to protocolnumber X5D052G, which incorporates by reference Northview StandardOperating Procedure 16D-07 and is on file in Northview PacificLaboratories, Inc. No amendments were made to the protocol.

Three New Zealand White rabbits were used. One day prior to dosing, thehair on each animal's back was removed with clippers. A one inch squaregauze patch containing a 0.5 mL volume of the test article was appliedto the shaved skin on each animal. The patch was held in place withsurgical tape. After application the trunk of each animal was wrappedwith gauze to prevent the animal from disturbing the test patch. Duringan exposure period of 4 hours, the animal was not restrained in any way.After the exposure period, the patch was removed and test articleresidues were gently rinsed off with a non-irritating solvent, water.The dosing sites were re-examined and scored 30-60 minutes, 24, 48, and72 hours after unwrapping. Additional scores were recorded 7 and 14 daysafter unwrapping to determine the time course of resolution for anyirritation that persisted.

The animals were observed for mortality, signs of ill health, orreaction to treatment.

Results

Signs of edema, erythema, and or eschar formation were scored for eachanimal according to the criteria in FIG. 4. The individual scores foredema and erythema are shown in FIG. 5. On the day of patch removal, allthree animals exhibited slight erythema (score of 1) at the 30-60 minutescoring and one animal (38383) had slight erythema to the 48 hourscoring. There were no irritation responses observed at the 72 hourobservation for any of the animals. All animals remained healthythroughout the study period. The test article was slightly irritating tothe skin of three test animals.

Example V Virucidal Efficacy of the Composition of the Invention

Method

The germicidal spray composition comprising 60% distilled water, 10%polysorbate 80, 5% orange valencia oil, 10% orange terpene oil, and 15%of a 35% Hydrogen peroxide was tested for virucidal efficacy.MICROBIOTEST INC. (the microbiology and virology laboratory 105Carpenter Drive, Sterling, Va. 20164) performed the tests from Feb. 10,2005 to Feb. 18, 2005 and all raw data, protocol, protocolmodifications, test material records, and the final report, are storedin the archives at MICROBIOTEST INC. (105 Carpenter Drive, Sterling, Va.20164) or at a controlled facility offsite. The Virucidal Efficacy testwas performed as detailed in the following protocol from MICROBIOTESTINC.:

Protocol for Virucidal Efficacy Test

Test conditions: Challenge virus: Avian Influenza A virus, Turkey/Wis/66 strain (H9N2), Charles River Laboratories; Host: Embryonatedchicken eggs, B&E Eggs; Active ingredient in test product: Hydrogenperoxide; Neutralizer used: Earle's Balanced Salt Solution containing0.3% Thioglycolic acid and 0.1% Catalase; Contact time: 10 minutes;Contact temperature: Ambient room temperature (22° C.); Dilution: Readyto use as received; Media and reagents:Earle's Balanced Salt Solution,Earle's Balanced Salt Solution containing 0.3% Thioglycolic acid and0.1% Catalase, Sephacryl S-1000, Chicken red blood cells, PhosphateBuffered Saline containing 0.5% fetal bovine serum, Phosphate BufferedSaline

Objective: The test is designed to substantiate virucidal effectivenessclaims for a product to be labeled as a virucide. It determines thepotential of the test agent to disinfect hard surfaces contaminated withviruses. The test is designed to stimulate consumer use and conforms toEPA Guidelines DIS/TSS-7, November 1981, and follows the procedureoutlined in the American Society for Test Materials (ASTM) test methoddesignated E 1053-97.

Testing conditions: Virus will be dried on a sterile glass Petri dish atambient temperature. Test agents specified by the sponsor in themiscellaneous section of the protocol will be used to treat the driedvirus according to the label claims. After a defined exposure period,the neutralized test agent-virus mixture will be scraped from thesurface, neutralized and assayed for the presence of infectious virus.

Materials: Test, control and reference substances will be supplied bythe sponsor of the study. The test agent will be tested as supplied bythe sponsor unless directed otherwise. All operations performed on thetest agent such as dilution or specialized storage conditions must bespecified by the sponsor before initiation of testing. The sponsorassures MICROBIOTEST testing facility management that the test agent hasbeen appropriately tested for identity, strength, purity, stability, anduniformity as applicable. MICROBIOTEST will retain all unused testagents for a period of at least three months after completion of thetest, then return them to the sponsor of the study or discard them in amanner that meets the approval of the safety officer.

Materials supplied by MICROBIOTEST, including, but not limited to: 1)Challenge virus (requested by the sponsor of the study): Avian Influenzavirus. 2) Host: Embryonated chicken eggs. 3) Laboratory equipment andsupplies. 4) Media and reagents: Media and reagents relevant to thevirus-host system and test agent being tested will be documented in thefirst project sheet and/or the data pack.

Test system identification: All Petri dishes, dilution tube racks, andhost-containing apparatus will be labeled with virus identification andproject number.

Experimental design: Procedures involved in performance of virucidalstudies are described a series of SOPs and logs that are maintained atMICROBIOTEST. The procedures used in different phases of the study willbe documented in the data pack.

Inoculum preparation: Viral stocks are purchased from reputable sourcesthat identify them by scientifically accepted methods and are propagatedat MICROBIOTEST. Records are maintained that demonstrate the origin ofthe virus. The virus stocks are stored at an ultra-low temperature.Frozen viral stocks will be thawed on the day of the test (fresh stockcultures may be used at the discretion of the Study Director).

Carrier preparation: An aliquot of 0.2 mL of stock virus will be spread,with a cell scraper, over an area of approximately 4 in² that has beenmarked on the underside of pre-sterilized Petri dishes. The virus willbe allowed to dry for 30 to 60 minutes at room temperature. The dryingtime and temperature will be recorded. One carrier will be prepared foreach test agent and the plate recovery control. One plate will beprepared for the neutralizer effectiveness control using an appropriatemedium.

Test agent preparation: The agent will be prepared according to thesponsor's directions or proposed label claims.

Test: After the carrier(s) are properly prepared, 2.0 mL of the testagent will be added. The plates will remain at the temperature and forthe time specified by the sponsor. Following the contact period, thetest agent will be neutralized with 2.0 mL of the appropriateneutralizing solution and the mixture will be scraped from the surfaceof the dish with a cell scraper. This will be considered approximately aone log₁₀ dilution. If columns are used, each sample will be loaded intoseparate pre-spun Sephacryl columns. Following passage through columns,the eluate will be removed aseptically and serially diluted. If columnsare not used, serial dilutions of neutralized virus-test agent mixturewill be prepared in using an appropriate diluent. For spray type agents,the agent will be used as the sponsor directs, the volume dispensed willbe measured and an equal volume of neutralizer will be used. Followingthe contact time, the procedure for processing the samples will be thesame as described earlier.

Viral host culture: Two-tenths mL of selected dilutions of theneutralized inoculum/disinfectant mixture will be inoculatedintra-allantoically in embryonic eggs and incubated for 5-7 days at37±2C. Four determinations will be recorded for each dilution of bothtests and controls. The eggs will be candled one-day post-inoculation oftest and control samples. All dead embryos will be discarded and thedata will be recorded. Following completion of the incubation period,the eggs will be candled and then kept at 2±2C overnight. Afterwards,the allantoic fluid will be harvested and kept at 2±2C until assay. Thesamples will be assayed for the presence of replicating virus usinghemagglutination assay following SOP 1006.11 (current version) and theresults will be recorded.

Controls

Neutralizer effectiveness (NE): This control will determine if residualactive ingredient is present after neutralization. One lot of the testagent will be used for the neutralizer effectiveness control. Thiscontrol will be processed exactly as the test procedure but instead ofviral inoculum, appropriate media will be added. Post neutralization, a1.0-mL sample will be divided into three portions, using two fortoxicity-related controls and the other for neutralizer effectiveness. A0.5 mL sample will be serially diluted, after which 100 μL of dilutedvirus will be added to each dilution and held for a period greater thanor equal to the contact time. Then these samples will be used toinoculate host embryos as described for the test procedure.

Toxicity (TX): The toxicity sample, acquired from the neutralizereffectiveness control, will be diluted and have no virus added. Selecteddilutions will be inoculated into the host and incubated in the samemanner as the rest of the test and control samples. These effects aredistinct from virus-specific cytopathic effects, which will be evidentin the stock titer and plate recovery control cultures.

Toxicity-related viral interference control: The test agent may not beeffective against the challenge virus yet be toxic to the host employedto detect its infectivity and may inhibit accurate interpretation of thetest. To determine the possibility of such interference by residualtoxic molecules, host treated with serially diluted neutralized testagent will be infected with a known number of infectious virions.Post-incubation they will be scored and compared with non-treatedinfected host cells control. This will rule out any possibility oftoxicity-related viral interference remaining in the neutralized testagent post-contact time.

Plate recovery (PRC): The carrier used will be prepared as the test. Avolume of an appropriate media equivalent to that of the test agent willbe added to the dried virus. Post contact time this sample will betreated as the test. This control will determine the relative loss invirus infectivity resulting from drying and neutralization alone. Theresults from this control will be compared with the test results toconfirm recovery of at least four log₁₀ of infectious virus followingdrying and neutralization. This titer will be compared with the titersof the test results to reach the acceptable test criteria. When samplesare required to pass through the Sephacryl columns, a column titercontrol (CTC) will be performed by assaying a portion of PRC beforepassing through the columns to determine the effect on infectious virustiter after passage through the columns.

Column titer control (CTC): This control will be performed to determineany effects the columns may have on infectious virus titer. It will onlybe performed if columns are used in the study. The sample for thiscontrol will be acquired from a portion of the PRC, prior to passingthrough the columns, and will be serially diluted in an appropriatemedia. It will then be processed in the same manner as the test.

Virus stock titer (VST): In order to verify the virus stock titer, alaliquot of the virus inoculum will be serially diluted in an appropriatemedia and processed, as well as assayed as described for the test.

(HVC) Eggs For Clarification: Four eggs will be inoculated with anappropriate media during the incubation phase of the study. This controlwill demonstrate that cells remain viable throughout the course of theassay period. In addition, it will confirm the sterility of the mediaemployed throughout the assay period.

Calculation: The 50% embryo infectious/lethal dose per mL (EID/ELD₅₀/mL)will be determined using the method of Reed and Muench, Am. J. of Hyg.1938, 27:493. The test results will be reported as the reduction of thevirus titer due to treatment with test agent expressed as log₁₀.

Product evaluation criteria: According to the regulatory agencies, thetest agent passes the test if there is complete inactivation of thevirus at all dilutions. When toxicity is evident, at least a three-logreduction in titer must be demonstrated beyond the toxic level. Testacceptance criteria: The test will be acceptable for evaluation of thetest results if the criteria listed below are satisfied. The studydirector may consider other causes that may affect test reliability andacceptance, a) The infectious virus recovered from the PRC control mustbe ≧4-log₁₀. b) Viral-induced toxicity must be distinguishable from testagent induced toxic effects.

Results

Results are presented in FIGS. 6-7. Avian Influenza virus was exposed tothe germicidal spray composition for 10 minutes at ambient roomtemperature (22° C.). The germicidal spray inactivated Avian Influenzavirus (FIG. 6), as no virus was detected after exposure. All controlsmet the criteria for a valid test (FIG. 7 a-c). Virus was not recoveredin the host viability control (FIG. 7 c) confirming media sterility andhost viability.

All references described herein are incorporated herein by reference.

1. A disinfectant composition comprising hydrogen peroxide (H₂O₂), orange terpene oil, orange valencia oil, a non-ionic emulsifier, and distilled or deionized water (H₂O).
 2. The composition of claim 1, wherein the orange terpene oil is present in the composition from 5% to 40% v/v, the orange valencia oil is present in the composition from 5% to 40% v/v, the non-ionic emulsifier is present in the composition from 5% to 50% v/v, the distilled or deionized H₂O is present in the composition from 5% to 80% v/v and the hydrogen peroxide is present in the composition from 1.5% to 8% w/v H₂O₂.
 3. The disinfectant composition of claim 1, wherein the non-ionic emulsifier is polysorbate
 80. 4. The composition of claim 1, wherein the composition comprises 5.25% w/v H₂O₂, 10% v/v orange terpene oil, 5% v/v orange valencia oil, 10% v/v polysorbate 80, and 60% v/v distilled water.
 5. The disinfectant composition of claim 1, further comprising oil of rosemary.
 6. The disinfectant composition of claim 1, further comprising an antimicrobial agent.
 7. The disinfectant composition of claim 1, further comprising a surfactant.
 8. The composition of claim 1, wherein the composition is formulated as a gel, or a spray, or a paste, or a disinfecting wipe.
 9. A method for disinfecting a surface comprising contacting a surface with the disinfectant composition of claim
 1. 10. A method for reducing the number of mold spores on a surface comprising contacting the mold spores with a disinfectant composition of claim
 1. 